One-pot multi-enzyme (OPME) chemoenzymatic synthesis of sialyl-Tn-MUC1 and sialyl-T-MUC1 glycopeptides containing natural or non-natural sialic acid

Article abstract of DOI:10.1016/j.bmc.2013.02.040

A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltran

Full text of DOI:10.1016/j.bmc.2013.02.040

Bioorganic & Medicinal Chemistry 21 (2013) 4778–4785
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
One-pot multi-enzyme (OPME) chemoenzymatic synthesis of
sialyl-Tn-MUC1 and sialyl-T-MUC1 glycopeptides containing natural
or non-natural sialic acid
Hamed Malekan a, Gabriel Fung b, Vireak Thon a, , Zahra Khedri a,à, Hai Yu a, Jingyao Qu a, Yanhong Li a,
Li Ding a,§, Kit S. Lam b, Xi Chen a,
⇑
a Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA
b Department of Biochemistry and Molecular Medicine, University of California, 2700 Stockton Blvd, Sacramento, CA 95817, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 7 March 2013
A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic
acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multien-
zyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 50-monophosphate-
sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine
50-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification
allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A
Keywords:
Carbohydrate
Chemoenzymatic synthesis
Enzymatic synthesis
Glycopeptide
T antigen
Campylobacter jejuni b1–3GalT (CjCgtB
one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1.
Sialylation of T-MUC1 using Pasteurella multocida 2–3-sialyltransferase 3 (PmST3) with Neisseria menin-
D30-His6) mutant has been found to catalyze the transfer of
a
Tn antigen
gitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced
ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates.
Ó 2013 Elsevier Ltd. All rights reserved.
ST antigen
STn antigen
1. Introduction
with many types of cancers. Aberrant expression of truncated
O-GalNAc glycans including Tn (GalNAc
3GalNAc Ser/Thr), and Thomsen-Friedenreich (TF or T-antigen,
Galb1–3GalNAc
aSer/Thr), STn (Siaa2–
Mucins are a family of high molecular weight extensively O-gly-
cosylated membrane-bound or secreted proteins produced by epi-
thelial tissues. They have various numbers of peptide tandem
repeats of diverse lengths and are involved in the protection and
lubrication of epithelia surfaces,1 signaling transduction regulation,
bacterial and viral binding, and are associated with embryogenesis,
carcinogenesis, and cancer metastasis.2 Among at least 19 mucins
discovered to date,3 mucin 1 (MUC1) is the most extensively stud-
ied one. Human MUC1 is a transmembrane-bound mucin whose
extracellular region contains 25–125 of 20-amino acid tandem
repeat (GSTAPPAHGVTSAPDTRPAP).4 The O-glycans of MUC1
expressed on normal epithelial cell surfaces are commonly core
a
a
Ser/Thr) antigens on mucin MUC1 are associated
with epithelial cancers.6–8 For example, STn-MUC1 has been found
on up to 80% of breast and ovarian cancer cells. Its expression is
associated with poor prognosis and is related to metastasis.9 Sialy-
lated T (ST, Siaa2–3 Galb1–3GalNAcaSer/Thr) antigen has also
found in numerous tumor tissues and cancer cell lines.10–12
Tn- and STn-MUC1 antigens have both been investigated as cancer
vaccine candidates. The STn-MUC1 vaccines studied,9,13–15 how-
ever, contain exclusively N-acetylneuraminic acid (Neu5Ac), the
most common sialic acid form. N-Glycolylneuraminic acid
(Neu5Gc), a sialic acid form commonly found in animals but cannot
be synthesized in humans due to the lack of an active CMP-Neu5Ac
hydroxylase (CMAH)16 and the absent of an alternative biosynthetic
pathway,17 has been found to be overexpressed on cancer cells by
metabolic incorporation.18–20 Low levels of circulating anti-Neu5Gc
antibodies in human can facilitate tumor progression via chronic
inflammation20,21 while high level of anti-Neu5Gc antibodies can
be used to kill cancer cells.22 Using a glycans microarray containing
a library of sialosides, elevated levels of antibodies against STn anti-
gen containing Neu5Gc (Neu5Gc-STn) have been recently found in
breast, prostate, ovary, lung, and endometrial cancer patients.22 The
2-type (Galb1–3[GlcNAcb1–6]GalNAcaSer/Thr) with extended N-
acetyllactosamine (LacNAc).5 Overexpression of MUC1 is associated
⇑
Corresponding author. Tel.: +1 530 754 6037; fax: +1 530 752 8995.
E-mail address: chen@chem.ucdavis.edu (X. Chen).
Current address: Department of Chemistry, Georgia State University, Atlanta, GA
30302, USA.
à
Current address: Department of Pharmaceutical Chemistry, University of Kansas,
Lawrence, KS 66047, USA.
§
Current address: College of Life Sciences, Northwest University, Xian, Shaanxi,
China.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.040

H. Malekan et al. / Bioorg. Med. Chem. 21 (2013) 4778–4785
4779
cancer-associated Neu5Gc-STn and anti-Neu5Gc-STn antibodies
can be potentially applied for cancer vaccine and immunotherapeu-
tic studies.
Metabolic engineering-based incorporation of C5-modified
non-natural sialic acids to cell surface glycoconjugates, combined
with vaccination using tumor associated carbohydrate antigen
(GM3, polysialic acid, or STn) analogs containing the same terminal
non-natural sialic acid,23–26 has been shown to be effective in elic-
iting specific antibodies for immunotherapy.
in one and a half hours by adding 5 equiv of each amino acid, HOBt,
and DIC coupling reagents except for GalNAc Ser (1.5 equiv)
a
which was carried out for 8 h. A dabsyl chromophore was intro-
duced as a UV/Vis probe to the N-terminus of the nonapeptide to
facilitate high-performance liquid chromatography (HPLC) analy-
sis.32 The resulting glycopeptide was released from the solid sup-
port by TFA treatment. Removal of the O-acetyl groups on the
GalNAc for the formation of target APG(GalNAca)STAPPA was
achieved by treatment with an aqueous solution of NaOH. To min-
Most anti-MUC1, anti-Tn-MUC1, or anti-STn-MUC1 antibodies
target the PDTRP region of MUC1.5,27 Patients with antibodies to
the GSTA region of MUC1 have also been identified. Recently,
monoclonal antibodies specifically recognizing Tn/STn-antigens
linked to the GSTA region of the MUC1 tandem repeats have been
identified and are believed to have potential in diagnosis and ther-
apeutic applications.5,28
imize the base-catalyzed cleavage of the glycosidic bond of Gal-
NAcaSer by b-elimination during O-acetyl deprotection, closely
monitoring of the reaction and controlling the pH of the reaction
in the range of 8.5–9.5 were critical. The produced Tn-MUC1 glyco-
peptide APG(GalNAca)STAPPA was used as a starting materials for
one-pot multienzyme (OPME) synthesis of T-MUC1 glycopeptide
as well as STn- and ST-MUC1 glycopeptides containing different
sialic acid forms (Scheme 1).
Here we describe solid phase-supported synthesis of Tn-MUC1
GSTA region APG(GalNAca)STAPPA and the application of efficient
one-pot multienzyme (OPME) chemoenzymatic systems in high-
yield synthesis of T-MUC1 glycopeptide as well as STn- and ST-
MUC1 glycopeptides containing Neu5Ac and its C5- or C9-modified
sialic acid derivatives such as Neu5Gc, N-azidoacetylneuraminic
acid (Neu5AcN3), and 9-azido-9-deoxy-N-acetylneuraminic acid
(Neu5Ac9N3).
2.4. One-pot three-enzyme synthesis of STn-MUC1
glycopeptides APG(Siaa2–6GalNAca)STAPPA
Two bacterial a2–6-sialyltransferases including Photobacterium
damselae (Pd2,6ST)33 and Photobacterium sp. (Psp2,6ST)8
a
2–6-
sialyltransferases were tested in a one-pot three-enzyme
a2–6-
sialylation system containing the sialyltransferase, Escherichia coli
sialic acid aldolase (EcNanA),34 and Neisseria meningitidis CMP-sia-
lic acid synthetase (NmCSS)33 for the synthesis of STn-MUC1 glyco-
peptides from chemically synthesized Tn-MUC1 glycopeptide
2. Results and discussion
2.1. Cloning, expression, and purification of CjCgtB
mutant
D30-His6
(Scheme 2). Both
a2–6-sialyltransferases worked well and led to
high efficient sialylation with comparable yields. Due to its higher
expression level, Pd2, 6ST was used for preparative-scale synthesis
of STn-MUC1.
A C-His6-tagged C-terminal 30 amino acid residue truncated
Campylobacter jejuni OH4384 b1–3-galactosyltransferase
(CjCgtB
Donor hydrolysis activity for which water molecules compete
with acceptors for glycosylation is common to glycosyltransfer-
ase-catalyzed reactions (Scheme 2).35 In order to remain sufficient
amount of CMP-Sia in the one-pot three-enzyme chemoenzymatic
sialylation system shown in Scheme 2, CTP was added periodically
during the reaction to allow continuous generation of sialyltrans-
ferase donor CMP-Sia from sialic acid formed by the donor hydro-
lysis activity of the sialyltransferase. This strategy worked well and
allowed high-yield production of the desired product. In addition,
flash C18 cartridge purification of the reaction mixture to separate
glycosylated product and remaining acceptor from other compo-
nents followed by an additional OPME reaction allowed the com-
plete consumption of the acceptor. The combination of adding
CTP periodically and C18 cartridge purification followed by an
additional OPME reaction was successfully exploited for the syn-
thesis of STn-MUC1 containing different sialic acid forms including
natural Neu5Ac and Neu5Gc as well as non-natural Neu5AcN3 and
Neu5Ac9N3 in excellent yields (89–91%) (Scheme 1, step c). The
purity of the products was demonstrated by high-performance li-
quid chromatography (HPLC) chromatograms and high-resolution
mass spectrometry (HRMS) spectra (Fig. 2 and Supplementary
data).
D
30-His6) mutant (amino acid changes in the mutant are:
N26K, L68I, K151E, L227G, and E234D)29,30 was cloned using a syn-
thetic gene with codon optimized for Escherichia coli expression
systems (Fig. 1). Expression in Luria-Bertani (LB) medium with iso-
propyl-1-thio-b-D-galactopyranoside (IPTG, 0.1 mM) induction fol-
lowed by nickel–nitrilotriacetic acid-agarose (Ni2+–NTA-agarose)
affinity column routinely produced 20 mg of purified protein per li-
ter of bacterial cell culture with >95% purity.
2.2. Synthesis of per-O-acetylated GalNAc a-linked to Fmoc-
protected serine as a building block for solid phase
glycopeptide synthesis of Tn-MUC1
Per-O-acetylated GalNAca(Fmoc)Ser containing a fluorenylm-
ethyloxycarbonyl (Fmoc)-protected amino group was synthesized
as a key building block for the solid-phase-supported synthesis
of Tn-MUC1 nonapeptide APG(Per-OAc-GalNAc
a)STAPPA. For the
synthesis of the per-OAc-GalNAc (Fmoc)Ser building block, tri-
a
methylsilyl trifluoromethanesulfonate (TMSOTf) was used as the
promoter. Peracetylated 2-azido-2-deoxygalactosyl trichloroace-
timidate was used as the glycosyl donor to minimizing the forma-
tion of b-GalNAc linkage by neighboring group participation of
GalNAc-based donors. Upon the completion of glycosylation, the
2-azido group was easily converted to N-acetyl by zinc reduction
of azide to amine followed by N-acetyl protection using acetic
anhydride.31
2.5. One-pot four-enzyme synthesis of T-MUC1 glycopeptide
APG(Galb1–3GalNAca)STAPPA
To obtain T-MUC1 glycopeptide APG(Galb1–3GalNAc
two OPME systems were tested including a one-pot two-enzyme
system containing Bifidobacterium infantis -galactosyl-b1–3-N-
a)STAPPA,
2.3. Solid phase synthesis of Tn-MUC1 glycopeptide
D
APG(GalNAca)STAPPA
acetyl-D-hexosamine phosphorylase (BiGalHexNAcP) and E. coli
galactokinase (EcGalK),36 and a one-pot four-enzyme galactosyla-
tion system containing Campylobacter jejuni b1–3-galactosyltrans-
Nonapeptide APG(Per-OAc-GalNAc
a
)STAPPA containing per-O-
acetylated GalNAc was produced by solid phase peptide synthesis
(Scheme 1). Each peptide bond formation reaction was performed
ferase (CjCgtBD
30-His6) mutant,30 E. coli galactokinase (EcGalK),

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H. Malekan et al. / Bioorg. Med. Chem. 21 (2013) 4778–4785
Figure 1. Synthetic gene and protein sequences of full length CjCgtB mutant. Five mutation sites (N26K, L68I, K151E, L227G, and E234D) are in bold and underlined.
Truncated C-terminal sequence in CjCgtB 30-His6 mutant is italicized.
D
Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP),
and Pasteurella multocida inorganic pyrophosphatase (PmPpA).37
Although BiGalHexNAcP was shown to have broad acceptor
in 55% yield and Gal-T-MUC1 glycopeptide with an additional Gal
residue in 34% yield.
substrate specificity and can use GalNAc
a
Ser and GalNAc
aThr as
2.6. One-pot three-enzyme synthesis of ST-MUC1 glycopeptides
APG(Siaa2–3Galb1–3GalNAca)STAPPA
substrates for high-yield (91–92%) synthesis of T-antigens,36 the
Tn-MUC1 glycopeptide APG(GalNAca)STAPPA was not an acceptor
substrate for the enzyme.
Enzymatic synthesis of ST-MUC1 glycopeptides APG(Sia
3Galb1–3GalNAc )STAPPA containing different sialic acid forms
from T-MUC1 glycopeptide was achieved by a one-pot three-en-
zyme 2–3-sialylation system similar to that described in Scheme 2
for the synthesis of STn-MUC1 glycopeptides except that an 2–3-
sialyltransferase, instead of an 2–6-sialyltransferase, was used.
Among several bacterial 2–3-sialyltransferases and mutants with
good expression levels including Pasteurella multocida 2–3-sialyl-
a2–
CjCgtBD
30-His6 mutant in one-pot four-enzyme system
a
(Scheme 3) produced the desired T-MUC1 glycopeptide. Quite
interestingly, after adding one galactose (Gal) residue to the Tn-
MUC1 glycopeptide for the formation of T-MUC1 glycopeptide,
a
a
the CjCgtB
D
30-His6 mutant was able to add additional Gal residues
a
to T-MUC1 glycopeptide for the formation of poly-Gal-glycopep-
tides as demonstrated by extra peaks in HPLC chromatogram and
confirmed by HRMS analysis. The additional galactose residues
are mostly likely linked through b1–3-linkage but this needs to
be confirmed in the future by a larger scale preparation of the
products followed by NMR characterization of the product. OPME
galactosylation with periodic addition of ATP and UTP combined
with flash C18-cartridge purification allow the total consumption
of the Tn-MUC1 glycopeptide and produced T-MUC1 glycopeptide
a
a
transferase 1 (PmST1),38 PmST2,39 PmST3,40 and PmST1 mu-
tants,35,41 PmST3 was chosen due to its good expression level
and excellent acceptor substrate tolerance towards oligosaccha-
rides and glycolipids. ST-MUC1 glycopeptides containing Neu5Ac,
Neu5Ac9N3, Neu5Gc, and Neu5AcN3 were obtained in excellent
yields (85–89%) (Scheme 1). The purity of the products was dem-
onstrated by high-performance liquid chromatography (HPLC)

H. Malekan et al. / Bioorg. Med. Chem. 21 (2013) 4778–4785
4781
Rink Amide
AcO
AcO
OAc
O
MBHA Resin
Loading = 0.55 mmol/g
AcHN
O
NHFmoc
O
O
O
O
O
H
N
H
N
H
N
O
S
O
H
N
N
N
N
N
N
N
N
H
N
H
NH
N
H
O
O
Fmoc SPPS
a
O
O
O
O
O
O
O
Dabsyl
1
R1
R2
Yield (%)
Cleave from beads
De-protection
b
OH
R2
CO2
O
3
4
5
6
N-Acetyl
N-Acetyl
N-Glycolyl
OH
N3
91
92
89
90
- OH
OH
OH OH
O
O
R1
O
OH
HO
HO
HO
-Azidoacetyl OH
N
AcHN
AcHN
O
O
O
OPME
O
O
O
O
O
H
N
H
N
H
N
H
H
N
H
N
2-6-Sialylation
α
N
N
N
N
N
N
N
Dabsyl
N
H
N
H
NH
NH2
Dabsyl
N
H
N
H
NH
NH2
c
O
O
O
O
O
O
O
O
O
O
O
O
OH
d
OH
2
OPME
R1
R2
OH
N3
Yield (%)
1-3-Galactosylation
55%
β
8
N-Acetyl
N-Acetyl
87
88
85
89
9
OH
OH
OH
O
OH
OH
-OH
OH
O
OH
O
OH
R2
CO2
10
-Glycolyl
11 N-Azidoacetyl OH
OH
N
OH
O
HO
O
O
O
O
R1
OH
AcHN
OH
AcHN
HO
O
O
OPME
O
O
O
O
O
O
H
H
N
H
N
H
N
H
N
H
2-3-Sialylation
α
N
N
N
N
N
N
N
N
Dabsyl
N
N
H
NH
OH
NH2
Dabsyl
N
H
N
NH
OH
NH2
e
H
H
O
O
O
O
O
O
O
O
O
O
O
O
7
Scheme 1. Solid phase synthesis of Tn-MUC1 glycopeptide APG(GalNAca)STAPPA (1) and one-pot multienzyme (OPME) chemoenzymatic synthesis of T-MUC1 glycopeptide
as well as STn- and ST-MUC1 glycopeptides containing different sialic acid forms. Reagents and conditions: (a) Standard solid phase glycopeptide synthesis using Fmoc-amino
acid-OH, HOBt, DIC in DMF. Dabsyl group was introduced using dabsyl chloride in DIPEA/DMF; (b) cleavage of glycopeptide from beads using TFA/TIPS/H2O cocktail for 2 h
followed by de-O-acetylation using NaOH/MeOH (pH was controlled in the range of 8.5–9.5) for 16 h; (c) one-pot three-enzyme
Pd2,6ST in Tris–HCl buffer (100 mM, pH 8.5) containing MgCl2 (20 mM); (d) one-pot four-enzyme b1–3-galactosylation using EcGalK, BLUSP, PmPpA, and CjCgtB
mutant in Tris–HCl buffer (100 mM, pH 7.5) containing MgCl2 (10 mM) and MnCl2 (10 mM); (e) one-pot three-enzyme 2–3-sialylation using EcNanA, NmCSS, and PmST3 in
Tris–HCl buffer (100 mM, pH 8.5) containing MgCl2 (20 mM). Enzymes used: EcNanA, Escherichia coli sialic acid aldolase; NmCSS, Neisseria meningitidis CMP-sialic acid
synthetase; Pd2,6ST, Photobacterium damselae 2–6-sialyltransferase; EcGalK, Escherichia coli galactokinase; BLUSP, Bifidobacterium longum UDP-sugar pyrophosphorylase;
PmPpA, Pasteurella multocida inorganic pyrophosphatase; CjCgtB 30-His6 mutant, Campylobacter jejuni b1–3-galactosyltransferase mutant; PmST3, Pasteurella multocida
2–3-sialyltransferase 3.
a2–6-sialylation using EcNanA, NmCSS, and
D
30-His6
a
a
D
a
OH
OH
O
CTP PPi
Mg2+
NmCSS
HO
Pyruvate
EcNanA
ManNAc and
Derivatives
AcHN
O
Sia
CMP-Sia
S
G
T
P
P
A
A
Dabsyl
P
A
H2O
Tn-MUC1 glycopeptide
Flash C18-
Cartridge
Pd2,6ST
OH
Purification
R2
-
CO2
OH
HO
OH
O
R1
O
O
HO
AcHN
CMP
O
S
G
T
P
A
A
Dabsyl
P
A
P
STn-MUC1 glycopeptide
Scheme 2. Strategies for optimizing synthetic yields of the one-pot multienzyme (OPME) sialylation reactions by adding excess amount of CTP and removal of CMP
byproduct by flash C18 cartridge purification (OPME synthesis of STn-MUC glycopeptide is used as an example).
chromatograms and high-resolution mass spectrometry (HRMS)
spectra (Fig. 2 and Supplementary data). The lower yields obtained
for the synthesis of ST-MUC1 glycopeptides compared to STn-
MUC1 glycopeptides were most likely due to the lower amount
(10-fold less) of the starting material used which led to a larger
percentage of material lost during reaction and purification.
forms. Periodic addition of nucleoside triphosphates during OPME
reactions for the regeneration of sugar nucleotides was shown to
improve the yields of the enzymatic reactions. This can be used
as a general strategy for glycosylating glycopeptides and glycopro-
teins. Flash C18-cartridge purification can be used to further
improve the glycosylation yields of OPME reactions for
glycopeptides. A newly reported OPME UDP-Gal in situ generation
system was successfully used with a b1–3-galactosyltransferase
3. Conclusion
(CjCgtB
MUC1 glycopeptide. CjCgtB
D
30-His6 mutant) in one pot for efficient synthesis of T-
30-His6 mutant was shown to be able
D
Several OPME chemoenzymatic approaches were shown to be
highly effective in synthesizing T-MUC1 as well STn- and ST-
MUC1 glycopeptides containing natural and non-natural sialic acid
to add one or more Gal residues to Tn-MUC1 glycopeptide for the
formation of T-MUC1 and Gal-T-MUC1 glycopeptides. STn-MUC1

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H. Malekan et al. / Bioorg. Med. Chem. 21 (2013) 4778–4785
a
b
100
1257.5649
100
80
2
2
[M + H]+
80
60
40
1279.5408
[M + Na]+
3
20
0
60
4
1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
100
1419.6118
[M + H]+
40
7
80
60
40
5
20
1441.5918
[M + Na]+
6
20
0
0
0
2
4
6
8
10 12 14 16 18 20 22 24
1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
100
100
1562.6472
[M ]-
7
5
80
60
80
60
40
20
0
40
8
20
0
1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
9
100
1724.6964
[M]-
10
80
60
40
10
11
20
0
1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
0
2
4
6
8
10 12 14 16 18 20 22 24
m/z
Minutes
Figure 2. (a) High-performance liquid chromatography (HPLC) chromatograms and (b) representative high-resolution mass spectrometry (HRMS) spectra of purified Tn-
MUC1 (compound 2) as well as T-MUC1 (compound 7), STn-MUC1 (compounds 3–6 containing Neu5Ac, Neu5Ac9N3, Neu5Gc, and Neu5AcN3, respectively), and ST-MUC1
(compounds 8–11 containing Neu5Ac, Neu5Ac9N3, Neu5Gc, and Neu5AcN3, respectively) glycopeptides synthesized by one-pot multienzyme (OPME) systems.
2Pi
OH
OH
O
PmPpA
ADP
Mg2+
EcGalK
ATP
UTP PPi
HO
Mg2+
AcHN
O
Gal
Gal-1-P
UDP-Gal
BLUSP
S
G
T
P
P
A
A
P
Dabsyl
A
H2O
Tn-MUC1 glycopeptide
Flash C18-
Cartridge
Mn2+
CjCgtB 30-His6
mutant
Purification
OH OH
O
OH
OH
O
O
HO
AcHN
HO
O
UDP
S
G
T
P
A
A
Dabsyl
P
A
P
T-MUC1 glycopeptide
Scheme 3. One-pot four-enzyme galactosylation system for the synthesis of T-MUC1 glycopeptide from Tn-MUC1. Enzymes used: EcGalK, Escherichia coli galactokinase;
BLUSP, Bifidobacterium longum UDP-sugar pyrophosphorylase; PmPpA, Pasteurella multocida inorganic pyrophosphatase; CjCgtBD30-His6) mutant, Campylobacter jejuni b1–3-
galactosyltransferase mutant.
and ST-MUC1 glycopeptides containing different sialic acid forms
can be useful probes for diagnosis. They can also be vaccine candi-
dates for therapeutic applications.
acid agarose), QIAprep spin miniprep kit, and QIAEX II gel extraction
kit were from Qiagen (Valencia, CA). Herculase enhanced DNA
polymerase was from Stratagene (La Jolla, CA). Electrocompetent
E. coli DH5a cells and chemically competent E. coli BL21 (DE3) cells
were from Invitrogen (Carlsbad, CA). T4 DNA ligase, 1 kb DNA ladder,
and restriction enzymes were from Promega (Madison, WI). N,N0-
Diisopropylcarbodiimide (DIC), N-hydroxybenzotriazole (HOBt),
MBHA-Rink amide resin, and amino acids were purchased from
Chem-Impex International (Wood Dale, IL). Triisopropylsilane (TIPS)
and N,N-diisopropylethylamine (DIPEA) were purchased from Fisher
4. Experimental
4.1. Materials
Vector plasmidpET22b(+) was purchased from Novagen (EMD Bio-
sciences, Inc., Madison, WI). Ni2+–NTA agarose (nickel–nitrilotriacetic

H. Malekan et al. / Bioorg. Med. Chem. 21 (2013) 4778–4785
4783
Scientific (Chicago, IL). Dabsyl chloride was purchased from
AnaSpec (Fremont, CA). -N-Acetylmannosamine (ManNAc) was pur-
pH 7.5). The fractions containing the purified enzymes were col-
lected, dialyzed against Tris–HCl buffer (20 mM, pH 7.5) containing
10% glycerol and stored at 4 °C.
D
chased from Sigma (St. Louis, MO). N-Acetylmannosamine derivatives
including N-glycolylmannosamine (ManNGc),42 N-azidoacetylman-
nosamine (ManNAcN3),42 and 6-azido-6-deoxy-N-acetylmannos-
amine (ManNAc6N3)38 were chemically synthesized as
reported previously. All other materials were obtained from commer-
cial suppliers and used as received with or without further
purification.
4.3. General methods
HPLC analysis and purification were carried out using Shimadzu
LC-2010HT (analytical run) and Waters 2525 binary gradient mod-
ule (preparative run). HPLC columns used for analysis and purifica-
tion were reverse-phase C18 columns: Waters Spherisorb ODS-2
4.2. Cloning, expression, and purification of Campylobacter
jejuni CgtB
(250 ꢀ 4.6 mm i.d., 5
l
m particle size) and Waters XTerra OBD
(150 ꢀ 19 mm i.d., 5
l
m particle size), respectively. High resolu-
tion electrospray ionization (ESI) mass spectra were obtained in
positive and negative modes using Thermo Electron LTQ-Orbitrap
mass spectrometer. Thin-layer chromatography (TLC) was per-
formed on silica gel plates 60 GF254. Flash C18 cartridge purifica-
tions were performed using Discovery DSC-18 3 mL tubes.
Glycopeptides were concentrated and dried using Thermo Scien-
tific SAVANT ISS110 SpeedVap concentrator.
4.2.1. Cloning
A synthetic gene encoding Campylobacter jejuni CgtB mutant
(N26K, L68I, K151E, L227G, and E234D) with codons optimized
for E. coli expression (designed based on GenBank accession no.
AAF31770) was custom synthesized by Biomatik (Wilmington,
DE, USA) and provided in a pGH vector. It was cloned into
pET22b(+) vector for a C-His6-tagged C-terminal 30 amino acid res-
idue truncated fusion protein. The primers used were: forward pri-
4.4. Synthesis of per-O-acetylated GalNAc a-linked to Fmoc-
protected serine as a building block for solid phase
glycopeptide synthesis of Tn-MUC1
mer
50-GATCCATATGTTCAAAATTTCTATCATCCTGCCG-30
(NdeI
restriction site is underlined) and reverse primer 50-CAGCGTC-
GACCTTAATTTTGTAGATCTGAATATAC-30 (SalI restriction site is
underlined). Polymerase chain reactions (PCRs) for amplifying the
Synthesis of the Tn building block, per-OAc-GalNAca(Fmoc)Ser,
target gene were performed in a 50
plasmid DNA (10 ng), forward and reverse primers (0.2
l
L reaction mixture containing
M each),
L) of Her-
was carried out similar to that reported.36 Briefly, glycosylation of
2-azido-2-deoxygalactosyl trichloroacetimidate donor (400 mg,
l
l
1ꢀHerculase buffer, dNTP mixture (0.2 mM), and 5 U (1
844
group) protected serine (290 mg, 760
84 mol) as a promoter provided glycosylated product with a good
lmol) with Fmoc (at amino group) and t-butyl (at the carboxyl
culase-enhanced DNA polymerase. The reaction mixture was sub-
jected to 30 cycles of amplification at an annealing temperature
of 55 °C. The resulted PCR product was purified and double di-
gested with NdeI and SalI restriction enzymes. The purified and di-
gested PCR product was ligated with the predigested pET22b(+)
l
mol) using TMSOTf (20 L,
l
l
yield (469 mg, 89%). Subsequent conversion of the azido group to
an N-acetyl group using Zn/THF/HOAc/Ac2O (472 mg, 80%) and
removal of the t-butyl group using TFA/H2O produced the glycosyl-
ated Fmoc-protected amino acid building block per-OAc-Gal-
vector and transformed into electrocompetent E. coli DH5
a cells.
Selected clones were grown for minipreps and characterized by
restriction mapping. DNA sequencing was performed by the Davis
Sequencing Facility in the University of California-Davis.
NAca(Fmoc)Ser in quantitative yield (442 mg, 100%).
4.5. Solid phase synthesis of Tn-MUC1 glycopeptide
APG(GalNAc )STAPPA
a
4.2.2. Expression
Positive plasmids were selected and transformed into E. coli
BL21 (DE3) chemical competent cells. The plasmid-bearing E. coli
strains were cultured in LB-rich medium (10 g/L tryptone, 5 g/L
yeast extract, and 10 g/L NaCl) supplemented with ampicillin
Tn-MUC1 glycopeptide APG(Per-OAc-GalNAca)STAPPA contain-
ing per-O-acetylated GalNAc was synthesized on Rink Amide-
MBHA resin (100 mg, 0.55 mmol/g) employing standard Fmoc solid
phase peptide synthetic procedures. The hydroxyl group of threo-
nine was protected by t-butyl group. Fmoc was removed from
the resin (Scheme 1) by treating the resin twice for 20 min each
time with 5 mL of 20% piperidine solution in DMF. Before and after
coupling of each amino acid, the resin was washed twice with 5 mL
of DMF, twice with 5 mL of methanol, and twice again with 5 mL of
DMF. Reactions were monitored by Kaiser Test before and after
each coupling. To add the first amino acid residue alanine (Ala)
(100
by inducing the E. coli culture with 0.1 mM of isopropyl-1-thio-b-
-galactopyranoside (IPTG) when OD600 nm reached 0.8–1.0 fol-
lg/mL). Overexpression of the target protein was achieved
D
lowed by incubating at 20 °C for 20 h with vigorous shaking at
250 rpm in a C25KC incubator shaker (New Brunswick Scientific,
Edison, NJ).
4.2.3. Purification
to the resin, Fmoc-Ala-OH (85 mg, 275
(42 mg, 275 mol, 5 equiv), and DIC (43
l
mol, 5 equiv), HOBt
The C-His6-tagged protein was purified from cell lysate. To ob-
tain the cell lysate, cell pellet harvested by centrifugation at
2683ꢀg for 2 h was resuspended in lysis buffer (pH 8.0, 100 mM
Tris–HCl containing 0.1% Triton X-100) (20 mL/L cell culture). Lyso-
l
lL, 275 mol, 5 equiv)
l
were dissolved in 5 mL of DMF and added to the resin in a capped
peptide synthesis column. The reaction was mixed constantly at
room temperature for more than 90 min using a Rotamix rotator
from Appropriate Technical Resources (ATR) until Kaiser Test as-
sured the completion of the reaction. The similar procedure was
applied for all peptide bond formation reactions with other non-
glycosylated amino acid residues. The coupling of per-OAc-Gal-
zyme (50 lg/mL) and DNaseI (3 lg/mL) were then added and the
mixture was incubated at 37 °C for 60 min with vigorous shaking.
Cell lysate was obtained by centrifugation at 11,270ꢀg for 30 min
as the supernatant. Purification of His-tagged proteins from the ly-
sate was achieved using a Ni2+-resin column. The column was pre-
equilibrated with 10 column volumes of binding buffer (5 mM
imidazole, 0.5 M NaCl, 50 mM Tris–HCl, pH 7.5) before the lysate
was loaded. After washing with 8 column volumes of binding buf-
fer and 10 column volumes of washing buffer (40 mM imidazole,
0.5 M NaCl, 50 mM Tris–HCl, pH 7.5), the protein was eluted with
an elute buffer (200 mM imidazole, 0.5 M NaCl, 50 mM Tris–HCl,
NAca(Fmoc)Ser was achieved by dissolving the building block
(70 mg, 84
lmol, 1.5 equiv), HOBt (13 mg, 84
lmol, 1.5 equiv),
and DIC (13
l
L, 84 mol, 1.5 equiv) in 5 mL of DMF and then mixed
l
with the resin. The reaction was carried out for more than 8 h until
Kaiser Test assured the completion of the reaction. To add a dabsyl
group, dabsyl chloride (36 mg, 111
lmol, 2 equiv) was added to
resin-bound peptide in 5 mL of DMF/DIPEA (95:5 by volume).

4784
H. Malekan et al. / Bioorg. Med. Chem. 21 (2013) 4778–4785
The resulting glycopeptide was then removed from the resin by
treatment with TFA/TIPS/H2O (95:2.5:2.5 by volume, 5 mL) for
2 h. The t-butyl protection group of threonine was simultaneously
removed during this step. The TFA was evaporated by rotary evap-
oration and cold diethyl ether (40 mL) was added to precipitate the
glycopeptide. The suspended mixture was transferred to a 50 mL
tube and centrifuged at 3835ꢀg for 5 min at 4 °C. The ether was
discarded by decanting. The glycopeptide was washed twice by
repeating the procedure (Crude yield = 65%). Deacetylation of the
O-acetylated glycopeptide was carried out by dissolving in
NaOH/MeOH (5 mL, pH 9.5) followed by incubation at room tem-
perature for 16 h. The deprotection process was monitored by
MALDI-TOF carefully. The pH was adjusted to 9.5 every hour by
4.8. One-pot three-enzyme synthesis of ST-MUC1 glycopeptides
APG(Sia 2–3Galb1–3GalNAc )STAPPA
a
a
T-MUC1 glycopeptide 7 (0.4 mg, 0.3 lmol, 1.0 equiv), sodium
pyruvate (5 equiv), and CTP (2 equiv) were dissolved in water in
a 0.5 mL centrifuge tube containing Tris–HCl buffer (100 mM, pH
8.5) and MgCl2 (20 mM). The resulting mixture was split equally
to 4 tubes and ManNAc or its derivative (1.5 equiv) was added to
each tube. After the addition of EcNanA (25
(30 g/mL), and PmST3 (50 g/mL), millipore water was added to
bring the volume to 80 L. The reaction and product purification
lg/mL), NmCSS
l
l
l
were carried out similarly to those described above for the synthe-
sis of STn-MUC1 glycopeptides.
adding NaOH to avoid the cleavage of labile a-GalNAc linkage un-
der acidic conditions caused by pH decrease due to acetic acid
formed. Upon completion, the reaction mixture was neutralized
using acidic Amberlite resin. After filtration and lyophilization,
the synthesized Tn glycopeptide were purified by reverse-phase
HPLC (Yield = 90%) and confirmed by high-resolution mass spec-
trometry (HRMS).
4.9. HRMS and HPLC analysis and purification of Tn-, T-, STn,
and ST MUC1
4.9.1. HPLC analysis and purification
The Tn-MUC1 glycopeptide (10 mg, retention time 13.6 min)
was purified by reverse-phase C18 column (Waters XTerra OBD,
5
l
m, 19 ꢀ 150 mm) with a flow rate of 10 mL/min and a gradient
elution of 0–50% of A from 0 to 30 min and monitored at 480 nm
[(A) CH3CN containing 0.1% TFA and (B) H2O containing 0.1%
TFA]. STn-MUC1 and ST-MUC1 glycopeptides were purified by re-
4.6. One-pot three-enzyme synthesis of STn-MUC1
glycopeptides APG(Siaa2–6GalNAca)STAPPA
verse-phase C18 column (Waters Spherisorb ODS, 2.5 lm,
Tn-MUC1 glycopeptide 2 (4 mg, 3
pyruvate (5 equiv), and CTP (2 equiv) were dissolved in water
and in 0.5 mL centrifuge tube containing Tris–HCl buffer
(100 mM, pH 8.5) and MgCl2 (20 mM). The resulting mixture was
split equally to 4 tubes and ManNAc or its derivative (1.5 equiv)
lmol, 1.0 equiv), sodium
4.6 ꢀ 250 mm) with a flow rate of 0.8 mL/min and a gradient elu-
tion of 30–50% of C from 0 to 17 min and monitored at 480 nm
[(C) CH3CN containing 0.1% formic acid and (D) H2O containing
0.1% formic acid]. HPLC purification of the T-MUC1 glycopeptide
was achieved similarly using 30–40% of C from 0 to 50 min. The
yields of OPME reactions were calculated by comparing the HPLC
peaks of the purified glycopeptides to the HPLC peak of a known
concentration of Tn-MUC1 glycopeptide standard. The values were
further confirmed using the molar extinction coefficient
a
was added to each tube. After the addition of EcNanA (25
mL), NmCSS (30 g/mL), and Pd2, 6ST (50 g/mL), ddH2O was
added to bring the volume of the reaction mixture to 300 L. The
lg/
l
l
l
reaction mixture was incubated at 37 °C for 6 h with shaking
(140 rpm). Additional CTP (1 equiv) was added and the reaction
was continued for 10 h. The product formation was monitored by
thin-layer chromatography (TLC) developed with EtOAc/MeOH/
H2O/HOAc (4:2:1:0.2) as the developing solvent. After TLC indica-
tion that the reaction reached 90% yield, the reaction mixture
was loaded to a C18 cartridge for flash purification. The cartridge
was washed six times with 1 mL of water each time and the glyco-
peptide was eluted with 2 mL of methanol and dried using Speed-
Vap concentrator. The obtained glycopeptide mixture was
subjected to another round of OPME reaction to allow the complete
consumption of the Tn-MUC1 glycopeptide acceptor. The resulting
STn-MUC1 glycopeptides were dissolved in a 1:1 mixture of
(e
= 32,000) of dabsyl measured at 480 nm. All glycopeptides were
analyzed by HPLC assays (Table 1) using an analytic C18 column
(Waters Spherisorb ODS, 2.5
l
m, 4.6 ꢀ 250 mm) with the same
conditions as for STn-MUC1 and ST-MUC1 purifications.
4.9.2. High-resolution LTQ-Orbitrap hybrid MS analysis
The mass spectrometric analysis of the obtained glycopeptides
was performed on LTQ-Orbitrap hybrid with nanospray source in
the positive mode for Tn-MUC1 and T-MUC1 glycopeptides 2 and
7. HRMS spectra for STn-MUC1 and ST-MUC1 glycopeptides 3–6
and 8–11 were obtained in the negative mode.
CH3CN/H2O (100 lL) and purified by reverse-phase HPLC column
as the final purification step.
Acknowledgments
This work was supported by NIH Grant R01HD065122, NSF
Grant CHE-1012511, and the Camille & Henry Dreyfus Foundation.
4.7. One-pot four-enzyme synthesis of T-MUC1 glycopeptide
APG(Galb1–3GalNAc
a)STAPPA
Table 1
Tn-MUC1 glycopeptide 2 (1 mg, 0.8
l
mol, 1.0 equiv), galactose
HPLC retention times (RT) and HRMS data of Tn-, STn-, T-, and ST-MUC1 glycopep-
tides synthesized
(1.5 equiv), ATP (2.0 equiv), and UTP (2.0 equiv) were dissolved in
water in a 0.5 mL centrifuge tube containing Tris–HCl buffer
(100 mM, pH 7.5), MgCl2 (10 mM), and MnCl2 (10 mM). After the
Glycopeptides
RT (min)
Exact mass (m/z)
Observed mass (m/z)
2
3
4
5
6
7
8
9
15.87
10.85
11.42
10.29
10.53
13.57
8.90
9.57
8.71
9.37
1257.5568a
1546.6377b
1571.6442b
1562.6326b
1587.6391b
1419.6097a
1708.6905b
1733.6970b
1724.6855b
1749.6919b
1257.5649a
1546.6521b
1571.6598b
1562.6472b
1587.6539b
1419.6118a
1708.7031b
1733.7067b
1724.6964b
1749.7059b
addition of EcGalK (40
mL), and CjCgtB 30-His6 mutant (55
to bring the total volume of the reaction mixture to 300
l
g/mL), BLUSP (50
g/mL), ddH2O was added
L. The
lg/mL), PmPpA (30 lg/
D
l
l
reaction mixture was incubated at 37 °C for 6 h with shaking at
1.5ꢀg. Additional ATP and CTP (equiv each) were added and the
reaction was continued for 10 h. The product formation was mon-
itored by TLC with EtOAc/MeOH/H2O/HOAc (4:2:1:0.2 by volume)
as the developing solvent. The C18 cartridge purification and the
repeat of OPME were carried out similar to those described above
for the synthesis of STn-MUC1 glycopeptides.
10
11
a
The masses given are for [M+H] (positive mode).
The masses given are for [MꢁH] (negative mode).
b

H. Malekan et al. / Bioorg. Med. Chem. 21 (2013) 4778–4785
4785
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Supplementary data
Supplementary data (high resolution mass spectrometry
(HRMS) spectra of glycopeptide products) associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2013.02.040.
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